The Effects of Acute Low-Volume HIIT and Aerobic Exercise on Leukocyte Count and Redox Status.

Oxidative stress is defined as a shift in the balance between the production of Reactive Oxygen and Nitrogen Species (RONS, free radicals) and the antioxidant defenses, favoring the oxidants (Halliwell, 2001). RONS are produced at all times as part of normal cell metabolism, as well as the environment. During exercise the production of free radicals can increase up to 20-fold in comparison to resting states and even up to 100-fold in active muscle groups (Jackson, 2008). While exercising, the high energy demands of active muscles cause an increase of oxygen ([O.sub.2]) consumption. This influx of [O.sub.2] in the mitochondrial electron transport chain makes for the large jumps in RONS production during exercise. If the amount of free radicals produced exceeds the amount that can be handled by the antioxidant defensive mechanisms, then we have what we call exercise-induced oxidative stress. It has been proposed that a single bout of exercise can result in an increase of oxidative stress and as a result in an upregulation of enzymatic antioxidant mechanisms (Michailidis et al. 2007). Redox changes are part of an exercise-induced inflammatory response that also includes the acute phase response, release of inflammatory mediators (cytokines), and immune cells mobilization and activation (Brenner et al., 1999; Nieman et al., 2012).

Regarding immune cells responses, an increase in leukocytes has been reported during and after short intense and longer submaximal exercise and resistance exercise (Brenner et al., 1999; Nieman et al., 2007; Natale et al., 2003), which may also trigger the increase in several cytokines (Nieman et al., 2006). Exercise-induced leukocytosis is characterized by a biphasic response in which the rise of leukocytes during and immediately after exercise is mainly due to an increase in both neutrophils and lymphocytes, while the delayed rise peaking several hours post-exercise is mainly due to circulating neutrophils (Hansen et al., 1991; McCarthy et al., 1991; Natale et al., 2003; Rowbottom and Green, 2000). Additionally, the magnitude of the response seems to be exercise intensity- and duration-related (Hansen et al., 1991; Rowbottom and Green, 2000).

Low volume high-intensity interval training (HIIT) has become popular since it has been shown to result in increased aerobic and anaerobic performance (Burgomaster et al. 2005; 2006). HIIT is characterized by a combination of intense anaerobic exercise with less intense recovery periods and is considered an excellent way to maximize a workout that is limited on time. Several studies have reported positive effects of HIIT on mitochondrial biogenesis (Little et al., 2010), glucose metabolism (Laursen and Jenkins, 2002), body composition (Zhang et al., 2017) and muscle and bone mass (Nybo et al., 2010). However, there are not many studies that compared redox status and inflammatory responses following acute HIIT and traditional continuous aerobic exercise (CET). A rise in lipid and protein oxidation indices as well as in antioxidant status markers have been reported following acute HIIT (Baker et al., 2004; Bloomer et al., 2006; Bogdanis et al., 2013). HIIT and weight training exercise have been reported to result in similar upregulation of redox status markers (Bloomer et al., 2006). On the other hand, a CrossFit workout (performed in an interval fashion and at a high intensity), resulted in lower protein carbonyl and total antioxidant capacity and higher lipid peroxides compared to a treadmill bout (Kliszczewicz et al., 2015). The duration of both exercise sessions was 20 minutes and the intensity of the treadmill exercise bout was 90% of maximal heart rate. It is evident that both of these exercise protocols were intense without differentiating the anaerobic from the aerobic component and therefore direct comparisons cannot be made. CET, usually characterized by low intensity, has been prescribed by exercise professionals as means to increase aerobic capacity and promote health and well-being. Increases in peak power outputs during exercise result in increased metabolic demands which in turn may compromise the integrity of skeletal muscle that could lead to early onset of fatigue and exhaustion and thus selection of exercise intensity should be done with caution in order to avoid undesirable outcomes.

As with redox status, not many studies have compared the effects of intense exercise and aerobic exercise on parameters related to immune cells changes such as white blood cell (WBC) count. In one of those, Mathes et al. (2017) found that a 4x30 sec all-out protocol resulted in significant perturbations of leukocytes, lymphocytes and neutrophils compared to high volume exercise (4x4 min at 90-95% V[O.sub.2max]). Furthermore, HIIT and moderate intensity training effectively depressed apoptosis and promoted autophagy in CD4 lymphocytes (Weng et al., 2013). However, the latter study was a training study and no firm conclusion can be drawn if the acute response of WBC-related variables is different between HIIT and CET. Nevertheless, such information is important when an exercise training program is developed and complete blood count is performed for health purposes. Therefore, the purpose of this investigation was to evaluate the effects of low-volume HIIT on WBC count and redox status and compare them with the effects of CET. The total time of involvement with exercise during HIIT was two times less compared to that during CET.

Methods

Participants

Twelve young males (22.4 + 0.5y) volunteered to participate in this two-trial, crossover investigation. In the present study, females were not included to avoid possible sex-dependent variability in the results, as it has been shown that sex accounts for different outcomes in exercise-induced inflammatory, oxidative stress, and cardiovascular parameters in both animals (Posa et al., 2013), and humans (Pepe et al., 2009; Wiecek et al., 2017). Additionally, the participants were about the same age, to avoid any age-related variability in the results of the study, since there is evidence that the response of oxidative stress biomarkers and anti-oxidants to exercise varies within different age groups (Barranco-Ruiz et al., 2017; Deli et al., 2017). Selection criteria included: a) absence of musculoskeletal and/or other health problems (cardiovascular disease or any other related health condition that could affect energy metabolism), b) no use of nutritional supplements, and medications before ([greater than or equal to]6 months) and during the study, c) participants were non-smokers. The anthropometric and physiological characteristics of participants are shown in Table 1. The procedures were conformed to the Helsinki declaration of 1975 and were approved by the Human Participants Committee of the local University (ref #:799/10-12-13).

Study design

The participants reported to the laboratory three times in total. All measurements took place in the morning (08.0010.00 a.m). At the first visit, anthropometric characteristics and maximal oxygen consumption (V[O.sub.2max]) were measured. Body mass was measured to the nearest 0.5 kg with the participants lightly dressed and barefoot (Beam Balance 710, Seca, UK) and standing height was measured to the nearest 0.5 cm (Stadiometer 208, Seca, UK). Percent body fat was calculated from 7 skinfold measures (average of 2 measurements at each site), using a Harpenden calliper (John Bull, St. Albans, UK). Maximal oxygen consumption (V[O.sub.2max]) was determined using a cycle ergometer (Monark 834E) test to exhaustion. The results of the initial maximal test were used to determine the exercise intensity of CET (70% V[O.sub.2max]). During the second and third visit (a washout period of one week between the second and third visit was included), the participants performed in random order either a CET protocol, or a HIIT protocol.

Maximal oxygen consumption

Each subject started pedaling at 70 revolutions per minute (rpm) with no additional workload for 150 s. Work rate (300g) was then added incrementally every 120 s with the intent of reaching the subject's V[O.sub.2max] within 6 to 12 min (Lundgren, et al. 2001). Criteria used to determine V[O.sub.2max] were: i) participants' exhaustion, ii) a

Submaximal exercise

Submaximal CET bout was performed on the cycle ergometer for 30 min. Exercise intensity was set at 70% of the participant's V[O.sub.2max]. The intensity of the exercise was checked through respiratory and heart rate measurements throughout the workout and adjustments were made so that participants were exercising at the prescribed intensity. In order to calculate the energy expenditure during the exercise, respiratory gas variables were measured for 30 min using a metabolic cart (Vmax29, Sensormedics, USA).

HIIT

The HIIT exercise bout involved the performance of four 30-sec sprints on a cycle ergometer (against a resistance of 0.375 kg/kg of body mass) interspersed with 4 min of recovery. In order to calculate the energy expenditure during the...